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Wingless signalling alters the levels, subcellular distribution and dynamics of Armadillo and E-cadherin in third instar larval wing imaginal discs.

Somorjai IM, Martinez-Arias A - PLoS ONE (2008)

Bottom Line: Surprisingly, DeltaNArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification.Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling.Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-Cadherin.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge, United Kingdom. ildiko.somorjai@obs-banyuls.fr

ABSTRACT

Background: Armadillo, the Drosophila orthologue of vertebrate ss-catenin, plays a dual role as the key effector of Wingless/Wnt1 signalling, and as a bridge between E-Cadherin and the actin cytoskeleton. In the absence of ligand, Armadillo is phosphorylated and targeted to the proteasome. Upon binding of Wg to its receptors, the "degradation complex" is inhibited; Armadillo is stabilised and enters the nucleus to transcribe targets.

Methodology/principal findings: Although the relationship between signalling and adhesion has been extensively studied, few in vivo data exist concerning how the "transcriptional" and "adhesive" pools of Armadillo are regulated to orchestrate development. We have therefore addressed how the subcellular distribution of Armadillo and its association with E-Cadherin change in larval wing imaginal discs, under wild type conditions and upon signalling. Using confocal microscopy, we show that Armadillo and E-Cadherin are spatio-temporally regulated during development, and that a punctate species becomes concentrated in a subapical compartment in response to Wingless. In order to further dissect this phenomenon, we overexpressed Armadillo mutants exhibiting different levels of activity and stability, but retaining E-Cadherin binding. Arm(S10) displaces endogenous Armadillo from the AJ and the basolateral membrane, while leaving E-Cadherin relatively undisturbed. Surprisingly, DeltaNArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification. However, only membrane-targeted Myr-DeltaNArm(1-155) produced comparable nuclear accumulation of Armadillo and signalling to Arm(S10). These experiments also highlighted a row of cells at the A/P boundary depleted of E-Cadherin at the AJ, but containing actin.

Conclusions/significance: Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling. Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-Cadherin.

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E-Cadherin levels change in the cytoplasm or basolateral membrane in response to changes in levels of endogenous Armadillo protein upon N-terminal deletion mutant overexpression.(A) E-Cadherin levels appear reduced in the domain of expression where endogenous Armadillo is excluded. Fewer puncta are also apparent relative to neighbouring wild type tissue upon overexpression of the ΔNArm1–155. (B) No change is evident in E-Cadherin levels when the membrane tethered Myr-ΔNArm1–155 form is overexpressed. (C) E-Cadherin levels are reduced in the domain of ArmΔCXM19 expression, and puncta are lacking. This is accompanied by an increase in the number of N27-postive puncta (red circles), representing endogenous or C-terminally truncated forms of Armadillo. There is also less E-cadherin associated with the anterior stripe, where endogenous Armadillo and E-cadherin perfectly colocalise under wild type conditions (red arrows). Note that in the wing disc in (C) only the dorsal aspect is shown, while (A) and (B) are show the intersection of the A/P and D/V boundaries.
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pone-0002893-g011: E-Cadherin levels change in the cytoplasm or basolateral membrane in response to changes in levels of endogenous Armadillo protein upon N-terminal deletion mutant overexpression.(A) E-Cadherin levels appear reduced in the domain of expression where endogenous Armadillo is excluded. Fewer puncta are also apparent relative to neighbouring wild type tissue upon overexpression of the ΔNArm1–155. (B) No change is evident in E-Cadherin levels when the membrane tethered Myr-ΔNArm1–155 form is overexpressed. (C) E-Cadherin levels are reduced in the domain of ArmΔCXM19 expression, and puncta are lacking. This is accompanied by an increase in the number of N27-postive puncta (red circles), representing endogenous or C-terminally truncated forms of Armadillo. There is also less E-cadherin associated with the anterior stripe, where endogenous Armadillo and E-cadherin perfectly colocalise under wild type conditions (red arrows). Note that in the wing disc in (C) only the dorsal aspect is shown, while (A) and (B) are show the intersection of the A/P and D/V boundaries.

Mentions: At the AJ, E-Cadherin levels appeared to be somewhat reduced when overexpressing ΔNArm1–155 (Figure 10A, blue channel). This was in contrast to ΔNArm1–128 (not shown), Myr-ΔNArm1–155 and ΔNArmXM19, which like ArmS10, did not appear to affect E-Cadherin (Figure 10B, C). E-Cadherin levels at the basolateral membrane, or possibly in the cytoplasm, (10%) were reduced when overexpressing ΔNArm1–155, particularly at the D/V boundary where the dppGAL4 domain intersects the Wingless signalling domain (Figure 11A, blue arrows). This slight reduction was not evident with Myr-ΔNArm1–155, where levels of E-Cadherin were either unchanged or slightly elevated (Figure 11B). In contrast, the ArmΔCXM19 construct caused a reduction of membrane E-Cadherin paralleling that of endogenous Armadillo where overexpressed, except at the anterior A/P stripe, where levels of ArmΔCXM19, and possibly endogenous Armadillo protein, appeared to be higher (Figure 11C). In addition, many Armadillo and E-Cadherin puncta were dissociated (red channel, circles). In combination, the discrepancy between the data from the AJ and the basolateral membrane suggested that the N- and C-termini play an important role in regulating E-Cadherin/Armadillo complex formation and subcellular distribution. Alternatively, association of the complex with other proteins might affect targeting of E-Cadherin and/or Armadillo to the membrane.


Wingless signalling alters the levels, subcellular distribution and dynamics of Armadillo and E-cadherin in third instar larval wing imaginal discs.

Somorjai IM, Martinez-Arias A - PLoS ONE (2008)

E-Cadherin levels change in the cytoplasm or basolateral membrane in response to changes in levels of endogenous Armadillo protein upon N-terminal deletion mutant overexpression.(A) E-Cadherin levels appear reduced in the domain of expression where endogenous Armadillo is excluded. Fewer puncta are also apparent relative to neighbouring wild type tissue upon overexpression of the ΔNArm1–155. (B) No change is evident in E-Cadherin levels when the membrane tethered Myr-ΔNArm1–155 form is overexpressed. (C) E-Cadherin levels are reduced in the domain of ArmΔCXM19 expression, and puncta are lacking. This is accompanied by an increase in the number of N27-postive puncta (red circles), representing endogenous or C-terminally truncated forms of Armadillo. There is also less E-cadherin associated with the anterior stripe, where endogenous Armadillo and E-cadherin perfectly colocalise under wild type conditions (red arrows). Note that in the wing disc in (C) only the dorsal aspect is shown, while (A) and (B) are show the intersection of the A/P and D/V boundaries.
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Related In: Results  -  Collection

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pone-0002893-g011: E-Cadherin levels change in the cytoplasm or basolateral membrane in response to changes in levels of endogenous Armadillo protein upon N-terminal deletion mutant overexpression.(A) E-Cadherin levels appear reduced in the domain of expression where endogenous Armadillo is excluded. Fewer puncta are also apparent relative to neighbouring wild type tissue upon overexpression of the ΔNArm1–155. (B) No change is evident in E-Cadherin levels when the membrane tethered Myr-ΔNArm1–155 form is overexpressed. (C) E-Cadherin levels are reduced in the domain of ArmΔCXM19 expression, and puncta are lacking. This is accompanied by an increase in the number of N27-postive puncta (red circles), representing endogenous or C-terminally truncated forms of Armadillo. There is also less E-cadherin associated with the anterior stripe, where endogenous Armadillo and E-cadherin perfectly colocalise under wild type conditions (red arrows). Note that in the wing disc in (C) only the dorsal aspect is shown, while (A) and (B) are show the intersection of the A/P and D/V boundaries.
Mentions: At the AJ, E-Cadherin levels appeared to be somewhat reduced when overexpressing ΔNArm1–155 (Figure 10A, blue channel). This was in contrast to ΔNArm1–128 (not shown), Myr-ΔNArm1–155 and ΔNArmXM19, which like ArmS10, did not appear to affect E-Cadherin (Figure 10B, C). E-Cadherin levels at the basolateral membrane, or possibly in the cytoplasm, (10%) were reduced when overexpressing ΔNArm1–155, particularly at the D/V boundary where the dppGAL4 domain intersects the Wingless signalling domain (Figure 11A, blue arrows). This slight reduction was not evident with Myr-ΔNArm1–155, where levels of E-Cadherin were either unchanged or slightly elevated (Figure 11B). In contrast, the ArmΔCXM19 construct caused a reduction of membrane E-Cadherin paralleling that of endogenous Armadillo where overexpressed, except at the anterior A/P stripe, where levels of ArmΔCXM19, and possibly endogenous Armadillo protein, appeared to be higher (Figure 11C). In addition, many Armadillo and E-Cadherin puncta were dissociated (red channel, circles). In combination, the discrepancy between the data from the AJ and the basolateral membrane suggested that the N- and C-termini play an important role in regulating E-Cadherin/Armadillo complex formation and subcellular distribution. Alternatively, association of the complex with other proteins might affect targeting of E-Cadherin and/or Armadillo to the membrane.

Bottom Line: Surprisingly, DeltaNArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification.Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling.Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-Cadherin.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge, United Kingdom. ildiko.somorjai@obs-banyuls.fr

ABSTRACT

Background: Armadillo, the Drosophila orthologue of vertebrate ss-catenin, plays a dual role as the key effector of Wingless/Wnt1 signalling, and as a bridge between E-Cadherin and the actin cytoskeleton. In the absence of ligand, Armadillo is phosphorylated and targeted to the proteasome. Upon binding of Wg to its receptors, the "degradation complex" is inhibited; Armadillo is stabilised and enters the nucleus to transcribe targets.

Methodology/principal findings: Although the relationship between signalling and adhesion has been extensively studied, few in vivo data exist concerning how the "transcriptional" and "adhesive" pools of Armadillo are regulated to orchestrate development. We have therefore addressed how the subcellular distribution of Armadillo and its association with E-Cadherin change in larval wing imaginal discs, under wild type conditions and upon signalling. Using confocal microscopy, we show that Armadillo and E-Cadherin are spatio-temporally regulated during development, and that a punctate species becomes concentrated in a subapical compartment in response to Wingless. In order to further dissect this phenomenon, we overexpressed Armadillo mutants exhibiting different levels of activity and stability, but retaining E-Cadherin binding. Arm(S10) displaces endogenous Armadillo from the AJ and the basolateral membrane, while leaving E-Cadherin relatively undisturbed. Surprisingly, DeltaNArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification. However, only membrane-targeted Myr-DeltaNArm(1-155) produced comparable nuclear accumulation of Armadillo and signalling to Arm(S10). These experiments also highlighted a row of cells at the A/P boundary depleted of E-Cadherin at the AJ, but containing actin.

Conclusions/significance: Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling. Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-Cadherin.

Show MeSH
Related in: MedlinePlus